GSM6729511: P2 SVZ, snRNAseq; Homo sapiens; RNA-Seq
Fresh frozen human brain tissue was minced, followed by addition of lysis buffer consisting of phosphate-buffered saline (PBS) containing 0.2% Triton X-100, 1X protease inhibitor cocktail (Roche), 1 mM DTT, 0.2 U/μl RNasin® ribonuclease inhibitor (Promega), and 2% BSA. After mechanical digestion, samples were incubated for 5 minutes on ice and centrifuged for 5 minutes at 500 x g at 4 °C. The resulting pellet, containing cell nuclei, was resuspended in PBS containing 1 mM EDTA, 0.2 U/μl RNasin®, and 2% BSA, and then filtered through a 20-μm pluriStrainer Mini (pluriSelect). DAPI was added and DAPI+ nuclei were collected by FACS in PBS containing 1 U/μl RNasin® and 5% BSA. The nuclei solution was concentrated by centrifugation for 15 minutes at 1000 x g at 4 °C and resuspended in PBS containing 0.2 U/μl RNasin® and 1% BSA, after which nuclei were counted. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.